5. The composition of claim 1, wherein the lipid-soluble molecule is
CoQ10.

6. The composition of claim 5, further comprising at least one
phospholipid.

7. The composition of claim 6, wherein the at least one phospholipid is
selected from the group consisting of phosphatidyl glycerol, phosphatidyl
inositol, phosphatidyl serine, phosphatidyl choline, and phosphatidyl
ethanolamine.

8. The composition of claim 5, further comprising at least one vitamin or
coenzyme selected from the group consisting of Vitamin A, Vitamin C,
Vitamin E, and NADH.

9. The composition of claim 5, further comprising at least one additional
component selected from the group consisting of DHA, triglycerides,
phosphatidic acids, ceramides, cerebrosides, sphingomyelins, and
cardiolipins.

10. The composition of claim 5, further comprising lecithin, soy or the
combination.

11. The composition of claim 1, wherein the angiogenin is isolated from a
biological fluid.

12. The composition of claim 11, wherein the biological fluid is selected
from the group consisting of colostrum, milk, whey, milk serum, blood,
plasma, and serum.

13. The composition of claim 11, wherein the biological fluid is obtained
from a mammal and wherein the mammal is selected from the group
consisting of humans, cows, buffalos, horses, sheep, pigs, and camels.

14. The composition of claim 1, wherein the interaction between the
angiogenin and the lipid-soluble molecule is non-covalent.

15. A method of treating a disease or disorder comprising administering
the composition of claim 1 to an individual in need thereof in an
effective amount.

17. The method of claim 15, wherein administration of the effective
amount is for supportive supplementation of long-term medications in the
management of hypertension, hyperlipidemia, diabetes, or chronic fatigue
syndrome in an individual in need thereof.

18. A method of preparing angiogenin complexed to a lipid-soluble
molecule comprising: thermally coating a fatty acid onto a quartz cover
slip or crystal resonator, immersing the coated fatty acid-coated cover
slip or crystal resonator in a solution comprising angiogenin, removing
the cover slip or crystal resonator comprising angiogenin complexed to
the fatty acid on the cover slip or crystal resonator from the solution,
rinsing the cover slip or crystal resonator comprising angiogenin
complexed to the fatty acid, and drying the cover slip or crystal
resonator comprising angiogenin complexed to the fatty acid.

19. The method of claim 18, wherein the fatty acid is stearic acid.

20. A method of preparing angiogenin complexed to a lipid-soluble
molecule comprising: (a) melting angiogenin with a composition comprising
phospholipid, (b) mixing sodium glycocholate with glycerol and heating,
(c) mixing (a) and (b), (d) sonicating the mixture formed in (c) (e)
optionally, preparing dilutions of the mixture formed in (d), (f)
combining the sonicated mixture with the lipid-soluble molecule.

21. The method of claim 20, wherein the lipid-soluble molecule is
selected from the group consisting of Coenzyme-Q10, Vitamin A and Vitamin
E.

Description:

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.
12/554,602, filed Sep. 4, 2009 which is a divisional of U.S. application
Ser. No. 11/734,729, filed Apr. 12, 2007, now U.S. Pat. No. 7,601,689.
Both application are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to protein stabilization, particularly
stabilization of angiogenin by immobilization on natural substrates which
includes but not limited to proteins, polysaccharides, lipids and
polyphenols.

[0004] 2. Description of the Related Art

[0005] Angiogenesis and vasculogenesis are two primary pathways in the
development and maintenance of mammalian health. The angiogenic role is
to supply and support tissue with ample vasculature, thus providing a
route of access for the transportation of essential nutrients, including
oxygen and the removal of metabolic waste in a sustained manner.
Angiogenesis is a strictly regulated, multi-step process that occurs
during normal physiology such as wound healing, pregnancy, and
development.

[0006] Angiogenin (ANG) has been shown to be a key mediating factor in the
underlying cascade of chemical events leading to angiogenesis, which
makes it a very important precursor molecule for both muscle development
and vascular generation. ANG is a 14-kDa, basic heparin-binding protein
and a member of the pancreatic ribonuclease (RNase) superfamily. ANG can
serve as a substrate for endothelial cell adhesion. ANG resembles
pancreatic RNase-A; their amino acid sequences are about 35% identical,
including the active site residues. An overview of the relationship of
ANG and other RNases of the super-family showed that their genes all are
in relative close proximity on human chromosome 14. However, human ANG
shows a weak ribonucleolytic activity (lower by 104 to
106-fold) despite of its potent angiogenic function. The actions of
ANG involve nearly all phases of angiogenesis (Strydom D J. Cell Mol.
Life Sci. 54:811-824, 1998; Acharya B et al., Proc. Natl. Acad. Sci. USA
91:2915-2919, 1994). When ANG was implanted into experimentally injured
menisci of New Zealand white rabbits, localized neovascularization
occurred in 52% of the treated animals as compared to 9% of controls
(King TV et al., J. Bone Joint Surg. Br. 73(4):587-590, 1991). Mutant ANG
proteins with enhanced angiogenic activity have also been reported (WO
89/09277). Site specific mutations in ANG resulted in mutant proteins
with increased RNase and angiogenic activities (U.S. Pat. No. 4,900,673).
Replacement of a specific section of ANG with a subsequence
characteristic of RNase unexpectedly resulted in a mutant ANG/RNase
hybrid with increased angiogenic activity (U.S. Pat. No. 5,286,487; U.S.
Pat. No. 5,270,204).

[0007] ANG (RNase type-4 and RNase type-5 forms) is an active secretory
protein found in milk. In cow's milk the concentrations are about 2 mg/L
for RNase 4 and between 1 and 8 mg/L for RNase 5 (Ye X Y, et al., Life
Sci. 67:2025-2032, 2000; Komolova G S, et al., Appl. Biochem. Microbiol.
38:199-204, 2002). ANG circulates in human plasma at a concentration of
about 0.3 μg/mL with a fast turnover rate and a half-life <5 min.
ANG can induce most of the events necessary for the formation of new
blood vessels. It binds avidly to endothelial cells and stimulates cell
migration and invasion. ANG promotes cell proliferation and
differentiation; mediates cell adhesion and activates cell associated
proteases; and also induces plasminogen activator and thereby, the
plasmin system promoting migration and tubular morphogenesis of
endothelial cells. Exogenous ANG is transported into the nucleus of
endothelial cells. The nuclear translocation results in accumulation of
the ANG in the nucleolus. Transportation of ANG from the cell surface
into the nucleus and subsequently to the nucleolus is critical for its
angiogenic activity. The import of ANG from the cytosol to the nucleus is
signal-dependent, carrier mediated and energy-dependent, active transport
process (Hu G F, et al., Proc. Natl. Acad. Sci. USA 94:2204-2209, 1997;
Moroianu J, et al., Proc. Natl. Acad. Sci. USA 91:1677-1681, 1994).

[0008] ANG is a potent inducer of neo-vascularization and the only
angiogenic molecule known to exhibit ribonucleolytic activity. Its
overall structure, as determined at 2.4 Å, is similar to that of
pancreatic RNase A, but it differs markedly in several distinct areas,
particularly the ribonucleolytic active center and the putative receptor
binding site, both of which are critically involved in biological
function. Most strikingly, the site that is spatially analogous to that
for pyrimidine binding in RNase A differs significantly in conformation
and is "obstructed" by Gln-117. Movement of this and adjacent residues
may be required for substrate binding to ANG and, hence, constitute a key
part of its mechanism of action (Acharya K R, et al., Proc. Natl. Acad.
Sci. USA 91:2915-2919, 1994; Russo N, et al., Proc. Natl. Acad. Sci. USA
91:2920-2924, 1994).

[0009] X-ray diffraction and mutagenesis results have shown that the
active site of the human protein is obstructed by Gln-117 and imply that
the C-terminal region of ANG must undergo a conformational rearrangement
to allow substrate binding and catalysis. Two residues of this region,
Ile-119 and Phe-120, make hydrophobic interactions with the remainder of
the protein and thereby help to keep Gln-117 in its obstructive position.
Furthermore, the suppression of activity by the intra-molecular
interactions of Ile-119 and Phe-120 is counter-balanced by an effect of
the adjacent residues, Arg-121, Arg-122 and Pro-123, which do not appear
to form contacts with the rest of the protein structure. They contribute
to enzymatic activity by constituting a peripheral sub-site for binding
polymeric substrates. These results reveal the nature of the
conformational change in human ANG and assign a key role to the
C-terminal region both in this process and in the regulation of human ANG
function (Russo N, et al., Proc. Natl. Acad. Sci. USA 93:3243-3247,
1996).

[0010] The pioneering work of Vallee and co-workers has paved the path in
the development of health applications for human ANG. U.S. Pat. No.
4,727,137 discloses therapeutic use of human ANG to promote the
development of hemo-vascular network, for example, to induce collateral
circulation following a heart attack, or to promote wound healing, for
example, in joints or other locations. This invention also describes
diagnostic applications of human ANG in screening for malignancies. U.S.
Pat. No. 4,952,404 describes healing of injured avascular tissue could be
promoted by applying human ANG in proximity to the injured tissue.

[0011] Besides an angiogenic factor, ANG has been used in the treatment of
viral infection such as HIV (WO 2004/106491A2). The RNase activity of ANG
seem to be an inhibitor of viral replication.

[0012] Activation of the receptor for ANG has been proposed as a method to
promote wound healing (WO 98/40487A1). A method of skin whitening by
applying a composition containing ANG has been described (U.S. Pat. No.
5,698,185). ANG was first isolated from human carcinoma cells and
subsequently from human plasma, bovine plasma, bovine milk, mouse,
rabbit, and pig sera and goat plasma (Maiti TK, et al., Prot. Pep. Lett.
9:283-288, 2002) and its use to diagnose cancer has been suggested (WO
02/25286).

[0013] However, the exploitation of human ANG polypeptide for
broad-spectrum human health-care (e.g., health supplementation, body
building, cosmetics, oral health, post-operative wound care) and animal
health applications (e.g., feed conversion for weight gains in
meat-yielding animals) is limited without a mass supply of the compound.
Such mass production of ANG requires an acceptable (preferably a
food-grade) raw material source and an effective large-scale purification
process for a high yield of ANG. Isolation of milk ANG from healthy dairy
animals could provide an answer to this limitation.

Bovine Milk ANG

[0014] Spik and co-workers described a method to isolate ANG from
mammalian milk. U.S. Pat. No. 5,171,845 discloses an extraction process
for ANG from cow milk consisting of a delipidation step by
centrifugation, chromatographic steps on SP-Sephadex® C50 and
S-Sepharose® columns, followed by a gel filtration step on
Bio-gel® P-30 column with a final fast protein liquid chromatography
(FPLC) step on Phenyl Superose® HR5/5 column. The protein yield was
estimated at 0.5 mg of ANG per liter of delipidated milk.

[0016] Bovine milk ANG is a single-chain protein of 125 amino acids; it
contains six cysteines and has a calculated molecular weight of 14,595.
Bovine milk ANG has 65% sequence homology with human plasma ANG and 34%
homology with bovine pancreatic RNase A. The three major active site
residues involved in the catalytic process, His-14, Lys-41 and His-115,
are conserved in the bovine milk ANG with ribonucleolytic activity
comparable to that of the human protein. Bovine milk ANG contains an
additional cell recognition tri-peptide Arg-Gly-Asp, which is not present
in the human ANG protein. In contrast to the human protein, the
N-terminus of bovine ANG is unblocked. Two regions, 6-22 and 65-75, are
highly conserved between human and bovine ANG proteins, but are
significantly different from those of the RNases, suggesting a possible
role in the molecules' biological activity. Bovine ANG has the following
sequence: NH2-Ala(1)-Gln-Asp-Asp-Tyr-Arg-Tyr-Ile-His-Phe(10)-Leu-Thr-
-Gln-His-Tyr-Asp-Ala-Lys-Pro-Lys(20)-Gly-Arg-Asn-Asp-Glu-Tyr-Cys-Phe-Asn-M-
et(30)-Met-Lys-Asn-Arg-Arg-Leu-Thr-Arg-Pro-Cys(40)-Lys-Asp-Arg-Asn-Thr-Phe-
-Ile-His-Gly-Asn(50)-Lys-Asn-Asp-Ile-Lys-Ala-Ile-Cys-Glu-Asp(60)-Arg-Asn-G-
ly-Gln-Pro-Tyr-Arg-Gly-Asp-Leu(70)-Arg-Ile-Ser-Lys-Ser-Glu-Phe-Gln-Ile-Thr-
(80)-Ile-Cys-Lys-His-Lys-Gly-Ser-Ser-Arg(90)-Pro-Pro-Cys-Arg-Tyr-Gly-Ala-T-
hr-Glu-Asp(100)-Ser-Arg-Val-Ile-Val-Val-Gly-Cys-Glu-Asn(110)-Gly-Leu-Pro-V-
al-His-Phe-Asp-Glu-Ser-Phe(120)-Ile-Thr-Pro-Arg-His-COOH (SEQ ID NO: 1).
Disulfide bonds link Cys(27)-Cys(82), Cys(40)-Cys(93), and
Cys(58)-Cys(108) (Maes P, et al., FEBS Lett. 241:41-45, 1988; Bond M D,
et al., Biochemistry 28:6110-6113, 1989).

[0017] Molecular dynamics simulation (MDS) studies showed marked
differences in the hydrogen-bonding patterns in the active site regions
of the human and bovine ANG systems. Furthermore, the positions of water
molecules identified in the crystal structures of human ANG significantly
differ from that of the bovine ANG. Positioning of the water molecules in
the protein structure play an important role in manifesting the subtle
functional differences between human and bovine ANG systems (Madhusudhan
M S, et al., Biopolymers 49:131-144, 1999).

[0018] Synthetic peptides corresponding to the C-terminal region of ANG
inhibit the enzymatic and biological activities of the molecule, while
peptides from the N-terminal region do not affect either activity.
Several C-terminal peptides also inhibit the nuclease activity of ANG
when tRNA is the substrate. Furthermore, peptide Ang(108-123) decreases
the neo-vascularization elicited by ANG in the chick chorioallantoic
membrane assay (Ryback S M, et al., Biochem. Biophys. Res. Commun.
162:535-543, 1989).

[0019] The mechanism of the angiogenic activity involves multiple
interactions of ANG with various molecules through specific regions on
its protein surface. The interactive molecules include heparin,
plasminogen, elastase, angiostatin, actin and most importantly a
170-kilodalton receptor on sub-confluent endothelial cells.

[0020] The interaction of ANG with heparin could protect the molecule from
protein cleavage by trypsin hydrolysis. A basic `triple` amino acid
cluster on ANG, Arg-31/Arg-32/Arg-33, has been identified as the heparin
binding site. Mutations of the triple cluster and of the Arg-70 residue
could decrease the binding affinity of ANG to heparin as well as its cell
adhesion property. However, a replacement of any other basic residues in
the polypeptide chain does not affect the heparin binding property of
ANG. The heparin binding site on ANG is outside the catalytic center.
Light scattering measurements on ANG-heparin mixtures suggest that a
single heparin chain (mass of 16.5 kDa) could interact with approximately
9 ANG molecules (Soncin F, et al., J. Biol. Chem. 272:9818-9824, 1997).

[0021] Several bio-molecules in milk and other exocrine secretions avidly
bind to heparan sulfate, the active constituent of "mucin" that overlay
the intestinal epithelia. The heparan sulfate interaction is generally
mediated by cationic domains located in the N-terminus region of such
bio-molecules. These immobilization processes facilitate retention of
biological compounds on epithelial surface and could possibly "activate"
these molecules for specific physiological functions, including their
internalization and bioavailability. Accordingly, heparan sulfate and its
analogues have a widespread application in chromatography as column
matrices, for purification and isolation of several milk compounds,
including ANG, lactoferrin, lactoperoxidase and other bioactive peptides.

[0022] U.S. Pat. No. 6,172,040 describes a method for immobilization of
milk lactoferrin (LF) on a galactose-rich polysaccharide (GRP) substrate,
which is analogous to the heparan sulfate. This immobilization process
involved the interaction of GRP with a highly cationic N-terminus domain
of LF, as expected. The immobilization process described in this
invention caused a significant increase in the antimicrobial activity of
LF, and also provided a structure-conformational stability to the protein
molecule.

[0023] The binding of ANG to heparan sulfate via its cationic N-terminus
domain, its mitogenic characteristics and occurrence in different
physiological milieu such as milk, plasma, other exocrine secretions and
tissue sites, is in striking proximity to LF. On a speculative basis, the
immobilization methods for LF, which are disclosed in U.S. Pat. No.
6,172,040, when adapted and applied to ANG, this angiogenic milk protein
has demonstrated a unique molecular and functional behavior.

[0025] Embodiments of the invention are directed to compositions which
include an isolated angiogenin non-covalently complexed to an isolated
naturally occurring substrate.

[0026] In preferred embodiments, the angiogenin is isolated from a
biological fluid. Preferably, the biological fluid is colostrum, milk,
whey, milk serum, blood, plasma or serum. Preferably, the biological
fluid is obtained from a mammal which is selected from humans, cows,
buffalos, horses, sheep, pigs and camels. In some embodiments, the mammal
is genetically modified.

[0028] In some preferred embodiments, the substrate includes
triglycerides.

[0029] In some preferred embodiments, the substrate includes a coenzyme
such as coenzyme-Q10 (ubiquinone) or NADH.

[0030] In some embodiments, the substrate includes a vitamin such as
vitamins A, C and/or E.

[0031] In some embodiments, the substrate includes a nucleic acid such as
single and double stranded DNA and RNA. In some embodiments, the
substrate includes a nucleotide such as ATP, CTP, GTP or TTP.

[0032] Embodiments of the invention are directed to methods of preparing
angiogenin complexes by mixing the angiogenin with a substrate in a
liquid medium. In preferred embodiments, the substrate includes a protein
such as transport proteins, subepithelial matrix proteins and
antimicrobial proteins. Preferably, the transport protein includes
lactoferrin, transferrin, ovo-transferrin (conalbumin), ceruloplasmin
and/or transfer factors. Preferably, the subepithelial matrix protein
includes fibronectin, fibrinogen, laminin, vitronectin, osteopontin,
native collagens and/or denatured collagen (gelatin). Preferably, the
antimicrobial protein is selected from peroxidases (lacto, myelo and
salivary forms) and lysozyme.

[0033] In some embodiments, the substrate includes triglycerides.

[0034] In preferred embodiments, the liquid medium comprises water.

[0035] Embodiments of the invention are directed to compositions that
include complexed angiogenin and native angiogenin. Preferably, the
composition includes complexed angiogenin and native angiogenin at a
ratio of 1:1 to 1:10. More preferably, the composition also includes a
buffer system, a physiological acceptable base and a salt. Preferably,
the buffer system is oxalic acid, ethylenediamine tetraacetic acid or
citric acid. Preferably, the physiological acceptable base is sodium
bicarbonate. Preferably, the salt is sodium chloride, potassium chloride
or calcium chloride.

[0036] Further aspects, features and advantages of this invention will
become apparent from the detailed description of the preferred
embodiments which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] These and other feature of this invention will now be described
with reference to the drawings of preferred embodiments which are
intended to illustrate and not to limit the invention.

[0038] FIG. 1: Isolation and Purification of ANG

[0039] Isolation of ANG using cation-exchange chromatography (UNO S6
column) with NaCl gradient (20% to 100% B) in 20 min. The time of
injection of ANG sample (1 mL of 0.2 mg/mL in 20 mM sodium phosphate
buffer pH 7.2) is indicated by the arrow. ANG eluted as two separate
peaks at 39 min. (38% Buffer B) and 44.5 min. (60% Buffer B)
respectively, from the time of injection. ANG was detected by monitoring
the absorbance at 214 nm using a UV-Vis detector.

[0040] FIG. 2: Biotinylated ANG-LF Interactions

[0041] Interaction of biotinylated LF and biotinylated ANG with
immobilized ANG ( ) and immobilized LF (.tangle-solidup.) was measured by
kinetic ELISA. ANG immobilized on a microtiter plate was incubated with
biotinylated LF, vice versa; LF immobilized on a microtiter plate was
incubated with biotinylated ANG, respectively. Unbound proteins were
removed by thorough washing with PBS-Tween buffer and the bound proteins
were subjected to an enzymatic reaction with avidin-alkaline phosphatase.
The dose dependence of rate of turnover of p-nitrophenyl phosphate
substrate by avidin-alkaline phosphatase on the concentration of
biotinylated protein indicated the interaction between LF and ANG.

[0042] FIG. 3. ANGex Detection by Turbidometry

[0043] Turbidity titration for the detection of ANGex in solution involved
measurement of A600 for a series of LF solutions to which increasing
amounts of ANG was added. The relative light scattering as measured by
the absorbance at 600 nm, plotted against the molar concentration ratio
of ANG to LF indicated non-linear increase in macromolecular size due to
complex formation. Inset shows linear increase of A280 for the same
series of solutions.

[0044] FIG. 4. Detection of ANGex Formation

[0045] Cation-exchange chromatogram is shown for the presence of ANGex,
eluted using 1 M NaCl gradient of (60-100% B in 80 min) and monitored by
the absorbance at 214 nm. (fdn)-LF (1 mg/mL) elutes as a single peak at
retention time of 84.6 min (Curve 1). The retention time of this peak
increases upon ANGex formation with 1 mg/mL (Curve 2) and 4 mg/mL (Curve
3) of ANG.

[0046] FIGS. 5A and B. Antioxidant Activity of ANG, LF and ANGex

[0047] Antioxidant activity was determined by FRAP assay with Vitamin C,
TROLOX and FeSO4 as standards, by following the increase in
absorbance at 593 nm with time. FIG. 5A shows the FRAP reaction kinetics
data for LF, ANG and ANGex. The concentrations of the three standards,
LF, ANG and ANGex were 10 mg/ml (125 μM for LF, 694 μM for ANG).
FIG. 5B compares the antioxidant efficiency (r) as measured by the change
in absorbance with the concentration of ANGex. Lines represent ANGex
formed at varying concentrations of ANG with 0 (∘), 2 ( ), 6
() and 10 mg/mL (.box-solid.) of LF. ANGex formed with 2 mg/mL of LF
exhibits the highest antioxidant efficiency with r=0.046.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0048] While the described embodiment represents the preferred embodiment
of the present invention, it is to be understood that modifications will
occur to those skilled in the art without departing from the spirit of
the invention. The scope of the invention is therefore to be determined
solely by the appended claims.

[0049] Suitable ANG can be isolated from dairy sources including
colostrum, milk, whey and milk serum from humans, cows, buffalos, horses,
sheep, pigs or camels. Additionally, ANG also can be purified from other
biological fluids from animals (e.g. blood), recombinant sources and
genetically-modified organisms (GMOs). Recombinant ANG may be
cloned-expressed in either prokaryotic or eukaryotic cellular systems.
The ANG is isolated by any conventional method, such as by filtration
methods, chromatography techniques using ion-exchanger, molecular-sieve
or affinity columns.

[0051] ANG supplementation supports vascular health and promotes repair of
damaged or clogged vascular tissue. However, in order to function
properly, the structure of ANG must be stabilized.

[0052] The activity of ANG, like the activity of most proteins, is highly
dependent on the three-dimensional or tertiary structure of the protein.
If the protein does not have the proper conformation its activity is
diminished or lost. ANG's instability limits it usefulness. Milieu
conditions such as metals (copper in particular), carbonic ions, salts,
pH and conductivity affect the angiogenic properties of ANG. In addition,
protein isolation procedures, storage, freezing-thawing, can adversely
affect the biofunctionality of ANG. Consequently, before ANG can be used
for commercial application, it would be expected to become denatured or
inactivated. (Soncin F, et al. Biochem Biophys Res Comm 236:604-610,
1997)

[0054] The attachment of ANG to the substrate may be non-covalent or
covalent binding. The interaction may be at the N-terminus, the
C-terminus or any molecular region or site of the ANG protein. In some
embodiments, ANG is attached covalently to a polysaccharide, preferably a
galactose-rich polysaccharide by covalent attachment to the N-terminus of
ANG. In other embodiments, the complex is formed by a non-covalent
association between ANG and a protein, lipid, nucleic acid, vitamin or
carbohydrate molecule. The basis of the association may be electrostatic
or by hydrophobic interaction or using bifunctional reagents.

[0055] ANG is immobilized on a naturally occurring substrate. Such
substrates include organic compounds, which attach to the N-terminus
domain of the ANG protein. Most preferably, the substrate is a
galactose-rich polysaccharide. Suitable galactose-rich polysaccharides
include galactose derivatives comprising galactose, anhydrogalactose,
2-O-methyl-galactose, and 4-O-methyl-galactose, among others. The GRP
substrates can be purchased or extracted from commercial agars by known
methods. Other suitable biologically active substrates include proteins,
such as collagen, denatured collagen (gelatin), fibronectin, and casein;
polysaccharides, such as mucin, heparan sulfates, carrageenan, and
cellulose; nucleic acids and their nucleotides, such as deoxyribonucleic
acid and adenosine triphosphate; and lipids such as triglycerides.

[0056] ANG is immobilized on the substrate using any suitable technique.
For example, ANG can be immobilized simply by mixing the ANG with the
biologically active substrate in a suitable medium, such as deionized
water. The immobilization process is dependent on the quality of the
substrate as well as the quality of the ANG. Consequently, the amount of
substrate and the amount of ANG to be used in the immobilization reaction
will depend, inter alia, on the choice of starting materials. The
immobilization technique and the amounts of substrate and ANG will be
readily determinable by a skilled artisan without undue experimentation.

[0057] ANG complexes could be formed with a second protein based on
functional association or a synergy that may enhance ANG function. The
ANG protein complexes could be formed by physical, charge and/or chemical
interactions. ANG and a protein substrate may be complexed together
directly or they may be complexed by means of an appropriate bifunctional
reagent. A non-covalent complex may be formed by means of electrostatic
interactions which may be enhanced by inclusion of appropriate buffers
and/or salts.

[0058] Embodiments of the invention include methods such as biotin-avidin
binding and disulfide bonding. The ANG polypeptide chain consists of 6
cysteine residues that form 3 disulfide bonds at 26-81, 39-92, and 57-107
residue positions. ANG may be labeled with biotin and mixed with a
substrate protein associated with avidin to form a complex by association
of the biotin with the avidin. Alternatively, the ANG may be attached to
avidin and the biotin complexed onto the substrate protein.

[0059] The formation of a complex may be confirmed by using
co-immunoprecipitation techniques. In preferred embodiments, the protein
substrate is from the group including, but not limited to transport
proteins, subepithelial matrix proteins and antimicrobial proteins.
Transport proteins include but are not limited to lactoferrin,
transferrin, ovo-transferrin (conalbumin), ceruloplasmin,
metallo-thionein and transfer factors. Subepithelial matrix proteins
include but are not limited to fibronectin, fibrinogen, laminin,
vitronectin, osteopontin, native collagens and denatured collagen
(gelatin). Antimicrobial proteins include but are not limited to
peroxidases (lacto, myelo and salivary forms) and lysozyme.

[0060] Embodiments of the invention are directed to ANG complexed with
lipids. ANG may be formed into a complex with CoQ-10 by physical and/or
chemical interactions. ANG and CoQ-10 may be complexed together directly
or they may be complexed by means of an appropriate bifunctional reagent.
A non-covalent complex may be formed by means of electrostatic
interactions which may be enhanced by inclusion of appropriate buffers
and/or salts.

[0061] In some embodiments, ANG is incorporated into anionic lipid films
by electrostatic interactions. ANG may be complexed into micro-emulsions.
Lipids used to form such films and micro-emulsions include triglycerides,
phospholipids including commercially available preparations such as
Phospholipid Lipoid S100 (Lipoid KG, Germany), lipophilic vitamins such
as Coenzyme-Q10, Vitamin A, Vitamin E and Ubiquinone. Other Vitamins
which complex with ANG include nicotinamide adenine dinucleotide (NADH)
and Ascorbic acid. More preferably, the phospholipid is one or more
selected from Docosahexaenoic acid (DHA), phosphatidyl glycerol,
phosphatidyl inositol, phosphatidyl serine, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidic acids, ceramides, cerebrosides,
sphingomyelins and cardiolipins.

[0062] Nucleic acid/nucleotide-based ANG complexes may be formed based
upon electrostatic interations between the positively charged ANG and the
negatively charged DNA or RNA. The formation of such complexes is
confirmed by Gel Mobility Shift Assay (Bading H. Nucleic Acid Research
16: 5241-5248, 1988).

[0063] In an embodiment of the present invention, ANG may be combined with
metal ions such as copper and zinc, preferably copper.

[0064] In some embodiments, ANG is used as an aqueous solution containing
a mixture of the ANGex and native ANG, where the concentration of the
mixture in the solution is from about 0.001 to about 2.5% wt/vol and the
ratio of ANGex to native ANG in the mixture is from about 1:1 to about
1:10, preferably about 1:1 to 1:5, and most preferably about 1:1. And in
some embodiments, the mixture contains about 1% wt/vol ANGex and about 1%
wt/vol native ANG.

[0065] In some embodiments, the aqueous solution further includes a buffer
system that contains a physiologically acceptable acid, such as oxalic
acid, ethylenediamine tetraacetic acid, and citric acid, preferably
citric acid, a physiologically acceptable base, preferably sodium
bicarbonate, and a physiologically acceptable salt, such as calcium
chloride, potassium chloride and sodium chloride, preferably sodium
chloride. The molar ranges of acid:base:salt is generally about 0.1 to
0.0001M (acid): 1 to 0.001M (base): 10 to 0.01M (salt); with 0.01-0.001M
(acid): 0.1 to 0.01M (base): 1 to 0.01M(salt), preferred; and 0.001M
(acid): 0.01M (base): 0.1M(salt), most preferred.

[0066] ANG useful in accordance with the present invention includes ANG
isolated from mammalian sources (humans, cows, sows, mares, transgenic
animals and the like), biological secretions such as serum, colostrum,
transitional milk, matured milk, milk in later lactation, and the like,
or processed products thereof such as skim milk and whey. Also useful is
recombinant ANG cloned-expressed in either prokaryotic and eukaryotic
cells. ANG is isolated by any conventional method, such as by
chromatography, ion-exchanger, molecular-sieve or affinity column. In a
preferred embodiment, ANG is isolated from plasma, serum, milk or a milk
product. Despite their low abundance, ANG is easily isolated from milk or
serum because it is more basic than other RNases. In a particularly
preferred embodiment, ANG is co-isolated with lactoferrin from milk or
milk product (Bond, et al., Biochemistry 27:6282-6287, 1988). The
ANG/lactoferrin isolate is then stabilized as described herein.

[0067] Without intending to be limited by a theory of operation, it is
believed that immobilization gives structural stability and
bio-functional specificity to ANG. It is expected that ANGex molecules
will demonstrate a molecular orientation similar to its orientation when
adhered to endothelial and fibroblast cells when forming the
extra-cellular matrix which is part of the angiogenic process. This
property facilitates the retention and carry-through of ANG.

[0068] In preferred embodiments, the ANGex is combined with native ANG.
The molecular ratio of ANGex versus native ANG is important in the
specificity, broad-spectrum activity, and molecular stability of both the
immobilized and the native ANG. Mixtures of ANGex and native ANG in a
ratio of from about 0.25:1 to about 1:10, preferably from about 1:1 to
about 1:2 of native ANG to ANGex, most preferably 1:1 ratio are
preferred.

[0069] Mixtures of ANGex and native ANG are formed by adding excess ANG to
the substrate. In a representative embodiment, from about 0.001% wt/vol
to about 2.5% wt/vol, preferably from about 0.5% wt/vol to about 2.0%
wt/vol, most preferably about 1% wt/vol of ANG is added to a solution
containing 0.01% wt/vol galactose-rich polysaccharide.

[0070] In a preferred embodiment, the aqueous solution is buffered with a
combination of a physiologically acceptable acid, such as oxalic acid,
ethylenediamine tetraacetic acid, or citric acid, preferably citric acid,
a physiologically acceptable base, preferably sodium bicarbonate, and a
physiologically acceptable salt such as calcium chloride, potassium
chloride or sodium chloride, preferably sodium chloride. The citrate and
bicarbonate ratio in the buffer is significant for co-coordinated metal
binding properties of ANG. The molar ranges of acid:base:salt is
typically about 0.1 to 0.0001M (acid): 1 to 0.001M (base): 10 to 0.01M
(salt); with 0.01-0.01M (acid): 0.1 to 0.01M (base): 1 to 0.1M(salt),
preferred; and 0.001M (acid): 0.01M (base): 0.1M(salt), most preferred.

[0071] ANGex may be substituted for ANG for any treatment for which ANG is
useful. ANGex brings the added benefits of increased stability for ANG as
an active agent. Because of the increased stability, ANGex has increased
residence time compared to ANG so that both dosage and frequency of
administration is less than with ANG.

[0072] ANGex is useful to improve cardiovascular function due to its well
known ability to promote formation of new blood vessels. In addition,
ANGex has antimicrobial and antioxidant properties as shown herein.

[0073] ANGex may be useful in treatment of cardiovascular disease,
gastrointestinal disorders, maintenance of bone and joint health,
maintenance of cognitive health, oropharyngeal health. ANGex may have
cosmetic uses such as hair growth and regeneration, anti-aging, reduction
of wrinkles and age spots, and skin rejuvenation. ANGex may be useful in
the treatment of acne and other skin disorders including folliculitis,
furunculosis, and IED. ANGex may be useful in post-operative recovery
including surgical wound repair/healing, tissue resurfacing, and plastic
surgery, both cosmetic and reconstructive. ANGex may be useful as a
coating on biomaterials including sutures, implants, indwelling catheters
and dental floss. ANGex may also be useful in veterinary practices and
for increasing weight gain in meat animals.

EXAMPLES

Example-1

Angiogenin (ANG) Preparations

[0074] Bovine milk (6% fat, 3% protein) was concentrated 2-3 fold, by
ultra-filtration through a polysulfonic membrane (pore size 30 kDa). ANG
was isolated from the ultra-filtrate by precipitation of proteins with
ammonium sulfate, followed by cation-exchange chromatography as
previously described (Fedorova TV, et al., Appl. Biochem. Microbiol.
38:193-196, 2002). Briefly, the protein precipitated with 60% ammonium
sulfate was dissolved in 0.01 M potassium phosphate buffer (pH 6.7) and
dialyzed against the same buffer at 4° C. for 36 h. The dialysate
was loaded on to a CM-cellulose 52 column (Serva, Germany) and 4-mL
fractions were collected. Fractions with A280≧0.1 were pooled and
dialyzed against 10 mM Tris-HC1 buffer (pH 8.3). The dialyzate was then
loaded on a CM-Toyopearl 650 S column (Tosoh, Japan) equilibrated with
0.05 M Tris-HCl buffer (pH 8.3). Column-bound proteins were eluted with a
linear gradient of KCl concentration in the same buffer at a flow rate of
0.5 mL/min. ANG-containing fractions were further pooled, dialyzed
against a 0.01 M potassium phosphate buffer (pH 7.0), and freeze-dried.

[0075] Furthermore, a second preparation of bovine milk ANG (mixture of
RNAse type-4 and -5 proteins) obtained from Tatua Nutritionals
(Morrinsville, New Zealand) was also used in the immobilization
experiments. This second preparation of ANG (initially enriched from skim
milk along with all other positively charged milk proteins) was isolated
by cation-exchange chromatography. The column bound proteins were eluted
step-wise by using various concentrations of salt (NaCl). The ANG-rich
fraction was de-salted and concentrated by ultra-filtration. This
ANG-rich preparation demonstrated >40% RNAse activity. For
experimental purposes, the ANG-rich fraction was further purified to a
higher level of purity, as follows: the ANG-rich fraction was run on a
second type of cation-exchange column and protein was eluted with
increasing concentrations of NaCl and by increasing the pH. This process
separated ANG into (RNAse 4)-rich and (RNAse 5)-rich fractions, which
were ultra-filtered and freeze-dried into powder form.

[0076] The purity and homogeneity of both lab-scale and pilot-scale ANG
preparations was assessed by cation-exchange chromatography. The sample
was loaded on to a UNO S6 column (Bio-Rad) equilibrated with 20 mM sodium
phosphate buffer at pH 7.2. After extensive washing with the same buffer,
ANG was eluted from the column by a gradient flow of 20 mM sodium
phosphate buffer containing 1 M sodium chloride (20 to 100% in 20 min).
Molecular weight of ANG (17 kDa) was confirmed by SDS-PAGE.

[0077] FIG. 1 depicts the isolation and purification of ANG using UNO S6
cation-exchange column. The time of injection of ANG sample (1 mL of 0.2
mg/mL in 20 mM sodium phosphate buffer pH 7.2) is indicated by the arrow.
ANG eluted as two separate peaks at 39 min. (38% Buffer B) and 44.5 min.
(60% Buffer B) respectively, from the time of injection. ANG was detected
by monitoring the absorbance at 214 nm using a UV-Vis detector.

[0079] ANG was biotinylated using biotin disulfide N-hydroxysuccinimide
ester (Sigma), which contains an active ester to react with the primary
amine of ANG. This reagent was dissolved in N,N-dimethylformamide at a
concentration of 25 mg/mL. ANG (20 mg) was dissolved in 2 mL of 0.1 M
sodium phosphate buffer, pH 7.6 to give a final concentration of 10
mg/mL. Reagent solution (volume: 0.4-mL volume) was slowly stir-mixed
with the ANG solution, such that a 15 molar excess of reagent is present
in the mixture. The mixture was gently agitated on a rocker for an hour
at room temperature. The biotinylated protein was separated from excess
reagent by gel filtration on a Sephadex G-25 column. The column was
equilibrated with 10 mM PBS containing 138 mM NaCl and 2.7 mM KCl at pH
7.4. The mixture was loaded on to the column and washed with few drops of
PBS. Biotinylated ANG (b-ANG) was eluted with PBS in 0.5 mL fractions
monitoring the A280 of the eluent to confirm the presence of b-ANG. All
the b-ANG fractions were pooled, its concentration was determined by
Bradford assay and the degree of biotinylation was estimated by
HABA/Avidin Assay.

[0081] Galactose-rich polysaccharide (GRP), extracted as a water-soluble
fraction from agar was used as the substrate for ANG immobilization. GRP
was prepared by adding 1-g of bacteriological agar (Difco) to 10-mL of
citrate-bicarbonate buffered saline [(CBS), pH 8.0; consisting of 1 mM
citric acid, 10 mM NaHCO3, and 100 mM NaCl]. After thorough mixing on a
vortex, the mixture was centrifuged for 2 min at 2500 rpm to obtain the
GRP supernatant. The clear GRP solution was carefully aspirated and used
as the substrate for ANG immobilization.

[0083] Accordingly, a carbohydrate substrate (i.e. GRP, carrageenan, GAG
or agarose) is thoroughly washed in distilled water, suspended in 0.2 M
HCl (2-3 mL/g moist gel) with gentle agitation. After incubation at
55° C. for 3 h, the gel suspension is neutralized with dibasic
sodium phosphate (0.2 M) solution, and washed with sodium phosphate (0.1
M) buffer at pH 7.2. The moist gel is filtered by gentle suction, weighed
and reconstituted to a known concentration in buffer (ca. 75 mg/mL). The
partially hydrolyzed gel is stored on ice and used within 3 hours of
preparation. ANG (protein concentration: 1 mg/mL) was dissolved in 0.1 M
sodium bicarbonate buffer with 0.5 M NaCl, pH 8.3. For immobilization, a
4 mL suspension containing 0.3 g moist gel in buffer solution was mixed
with ANG solution and 6 mL of 100 mg/mL cyanogen bromide solution in
Dioxan (150 mg of CNBr/mL of Sepharose®). The reaction mixture was
gently agitated at 25° C. for 1 h, loaded on to a column and the
gel (or beadlets) was extensively washed with coupling buffer (at least
5-6 column volumes) until no more amino groups (or excess protein) was
detected in the washings.

[0084] In another method, 0.3 g of moist gel was suspended in 4 mL of
buffer and was mixed with ANG solution and freshly prepared sodium
cyanoborohydride solution (80 mM). The mixture was agitated at room
temperature for 2 h and loaded on to a column and the gel (or beadlets)
was extensively washed with coupling buffer (at least 5-6 column volumes)
until no more amino groups (or excess protein) was detected in the
washings.

[0085] In order to block any remaining active groups, the gel is
transferred to 10 mL of 0.1 M Tris-HC1 buffer, pH 8.0 and allowed to
stand for 2 h. The gel (or beadlets) was washed again with 3 alternative
cycles of high and low pH buffers. Each wash cycle consisted of at least
5 column volumes of 0.1 M acetic acid/sodium acetate, pH 4.0 containing
0.5 M NaCl; followed by a wash with 5 column volumes of 0.1 M Tris-HCl,
pH 8.0 containing 0.5 M NaCl. This procedure ensured that no free ligand
(ANG) bound non-specifically to the gel.

[0087] For binding studies, a 1% LF substrate (cow milk protein isolate)
was prepared in 20 mM sodium acetate buffer (pH 4.0). A 1% LF solution
(volume: 0.1-mL) was added to each well of a microtiter plate (Corning
Costar® 3690) and allowed for an overnight surface adsorption at
4° C. The microplate was washed extensively with acetate buffer to
remove any unbound LF. A 0.5% Tween (in PBS) was added to each well to
block any unbound surface. Various concentrations (ranging 0 to 1 mg/mL)
of biotinylated ANG solution (in 20 mM PBS, pH 7.2) was added to
individual wells and further incubated at 4° C. overnight, to
achieve equilibrium. Any unbound ANG was removed by repeated washing with
PBS-Tween. Interactions between ANG and LF were measured by using
avidin-conjugated alkaline phosphatase reagent, followed by a color
reaction with the addition of p-nitrophenyl phosphate (PNP), a
chromatophore substrate. The rate of color development was measured at
405 nm, in a kinetic manner for 30 min. The rate of enzymatic hydrolysis
of PNP chromatophore as function of concentration of biotin-ANG was
plotted to measure the binding interactions.

[0088] FIG. 2 depicts the interactions of biotinylated LF with (fdn)-ANG
(subset-A), and biotinylated ANG with (fdn)-LF (subset-B), respectively,
as measured by kinetic ELISA. The rate of PNP substrate hydrolysis by
avidin-alkaline phosphatase, as measured by kinetic ELISA, indicated the
interaction of biotinylated protein(s) with (fdn)-ANG or -LF. Data shows
the formation of ANG-LF complexes in a dose-dependant manner.

Example 4

Preparation of ANGex Using Lipid-Based Substrates

[0089] Based on its cationic properties, ANG is incorporated into the
anionic lipid films and matrices by electrostatic interactions. A fatty
acid-like stearic acid (Octadecanoic acid C18H36O2) is thermally coated
under vacuum on a quartz cover slip or quartz crystal resonator of the
type used in Quartz Crystal Microbalance (QCM) technique. This process is
known to produce a film of few hundred angstrom thickness. The
stearate-coated quartz cover slip is immersed in 0.1% ANG solution (in 50
mM sodium phosphate buffer) at 4° C. for 30 min. The quartz cover
slip is removed from the ANG solution, washed thoroughly in deionized
water and dried under gentle flow of dry nitrogen gas.

Measurement of ANG Interaction with Lipid Film by QCM

[0090] Immobilization of ANG on a thermal evaporated fatty acid film was
monitored by measuring changes in the frequency of quartz crystals using
QCM instrument (Edwards FTM5 microbalance) at 1 Hz resolution. The
frequency changes are converted to mass loading using the Sauerbrey
equation. The quartz cover slip is also used for recording the UV
absorption spectrum of the film before and after the lipid immobilization
of ANG. The difference in the UV spectrum indicated the immobilization of
ANG on the lipid film (Sauerbrey G., Z. Phys. 155:206, 1959; Buttry DA,
et al., Chem. Rev. 92:1356-1379, 1992; Sastry M., Trends in Biotechnology
20:185-188, 2002).

[0091] ANG was immobilized and entrapped in the lipid matrix primarily by
electrostatic interactions. The use of charge interactions also enabled
"leaching-out" of the entrapped ANG when immersed in solution at pH 2.0.
Soft lipid matrix enabled the entrapment without significant distortion
of the tertiary structure of the ANG, therefore, the bio-functional
properties of the protein molecule is well preserved.

Example 5

Preparation of ANGex Using Coenzyme/Vitamin-Based Substrates

[0092] Coenzyme-Q10 (CoQ-10), due to its quinone structure, is extremely
lipophilic, soluble in ethanol and chloroform, but practically insoluble
in water. A detergent like sodium glycocholate along with sonication
generates a submicron-sized dispersion of CoQ-10 in phospholipid (from
soy bean). The detergent also inhibits re-crystallization of CoQ-10. This
dispersion has been used in the preparation of micro-emulsions to
incorporate ANG (Stojkovic M, et al., Biol. Reprod. 61:541-547, 1999).

[0093] Phospholipid Lipoid S100 (Lipoid KG, Germany) is a mixture of
phosphatidylcholine from fat-free soybean lecithin that consists mainly
of linoleic phosphatidylcholine. S100 was melted together with CoQ-10
(Alchem, India) at 65° C. Sodium glycocholate (NaGC) was dissolved
in distilled water containing 2.25% (w/w) glycerol. This mixture was
heated to 65° C. and mixed with the molten lipid phase. The hot
mixture was sonicated for 30 min. while maintaining a constant
temperature. This hot emulsion was sterile-filtered (0.22 μm) into
vials and allowed to cool at room temperature. Stabilizer dispersion
without CoQ-10 was also prepared in a similar manner. Calculated amounts
of the two dispersions were mixed to prepare dispersions containing
various concentrations of CoQ-10.

[0094] ANG stock solution was freshly prepared using 50 mM phosphate
buffer, pH 8.0, containing 0.2% sodium cholate. This solution was diluted
with 50 mM phosphate buffer, pH 7.4, containing 0.2% sodium cholate to a
protein concentration of 1 mg/mL. The ANG-CoQ-10 complexes were prepared
by mixing 500 μL aliquots of diluted ANG solution with 500 μL of
ubiquinone dispersions at various concentrations up to 100 μM. The
mixtures were kept in an incubator-shaker at 25 C for 1 h, before
characterization by antioxidant assay.

[0096] Ascorbic acid has pKa value of 4.2 and would complex with ANG. This
complex was prepared by mixing 500 μL of ANG (1 mg/mL) solution with
500 μL of ascorbate solutions of different concentrations in 50 mM
phosphate buffer, pH 7.4.

[0097] Vitamins A and E are also fat-soluble and their complex with ANG
was also prepared by dispersion in soy phospholipids, as described with
CoQ-10 complexes

Example 6

Preparation of ANGex Using Nucleic Acid/Nucleotide-Based Substrates

[0098] DNA, RNA and nucleotides are negatively charged due to the presence
of phosphate groups. These molecules form complexes via the positively
charged residues on basic proteins such as ANG. The characterization of
the affinity and shape of the ANG-nucleotide complex is performed using
Gel Mobility Shift assay (Bading H. Nucleic Acids Research 16:5241-5248,
1988). The DNA-binding assay using mobility-shift polyacrylamide gel
electrophoresis (PAGE) is based on the observation that protein/DNA
complexes migrate through polyacrylamide gels more slowly than unbound
DNA fragments.

[0099] ATP-protein complex was formed by the treatment of the ANG with ATP
(Sigma). ATP (10 μmoles) was incubated with ANG (5 mg) and 40 μM
Tris-acetate buffer, pH 7.0, in a total volume of 1 mL for 10 min at room
temperature. The complex was centrifuged, washed, and the ATP:Protein
ratio determined (Richardson SH, et al., Proc. Nati. Acad. Sci. USA
50:821-827, 1963).

Example 7

Preparation of [ANGex]+[(fdn)-ANG] Mixtures

[0100] The molecular ratio of ANGex versus (fdn)-ANG is important for the
specificity, multi-functionality and molecular stability of both the
immobilized and the native ANG proteins. Mixtures of ANGex and (fdn)-ANG
are formed by adding excess (fdn)-ANG during the immobilization,
conjugation or complexation process. Mixtures of ANGex and (fdn)-ANG in a
ratio of from about 100:1 to about 1:100, preferably from about 10:1 to
about 1:10, most preferably 1:1 ratio have been found to provide the
optimum stability and functionality.

Example 8

Detection of ANGex

[0101] The formation of ANGex was confirmed by a turbidity titration
method, commonly used to study formation of several macromolecular
complexes such as protein-DNA and protein-polyelectrolyte complexes.
During formation of ANGex (i.e. ANG+LF complex), the interaction is
accompanied by an increase in the macromolecular size that results in the
increase of incident light scattering. The relative light scattering is
quantified by spectral analysis as apparent absorbance at 600 nm, while
subtracting any interfering absorbance from the sample or buffer (Zhou Y,
et al., Biophys Chem 107:273-281, 2004; Xia J, et al., Langmuir
9:2015-2019, 1993).

[0102] FIG. 3 shows the detection and measurement of ANGex by turbidity
titration method. The assay is based on measurement of A600 for a
series of LF solutions to which increasing amounts of ANG was added.
ANGex (ANG+LF complex) was prepared in a series of samples with varying
amounts of ANG (0 to 25 μM) with LF (5 μM) solution in 20 mM
phosphate buffer. The protein absorbance at 280 nm showed a linear
increase with the molar ratio [ANG]/[LF] of the complex forming proteins.
The relative light scattering as measured by the absorbance at 600 nm,
plotted against the molar concentration ratio of ANG to LF indicated
non-linear increase in macromolecular size due to ANGex complex
formation. (Inset) shows linear increase in A280 absorbance for the
above series of solutions.

Isolation of ANGex

[0103] ANGex with carbohydrate or protein substrates are isolated by
size-exclusion and/or ion-exchange chromatography. Due to larger size,
ANGex is eluted earlier than the free proteins or free carbohydrates on
the size-exclusion column. A size-exclusion column (TSKgel G4000PW, Tosoh
Biosep, Japan) was equilibrated with 20 mM sodium phosphate buffer, pH
7.2. The ANGex solution was injected into to the column and subsequently
eluted with sodium phosphate buffer at a flow rate of 0.5 mL/min.

[0104] During isolation with cation exchange column (UNO-S; Bio-Rad), a
solution containing ANGex was applied to a cation exchange column which
was equilibrated with 20 mM sodium phosphate buffer (pH 7.2). A flow rate
for the buffer was continued at 1 mL/min for 30 min to ensure removal of
unbound chemical residues from the ANGex solution. The bound ANGex was
eluted by flow of salt gradient (1 M NaCl in phosphate buffer). In the
case of ANG-LF complex, free proteins and the complex were eluted as
separate peaks at distinct salt concentrations. However, for ANG-GAG
complex, a combination of a cation-exchange and an anion exchange columns
were used in series, since such complex shows residual negative charge
from the GAG component and binds to anion-exchange column along with free
GAG. On the other hand, the free protein due to its positive charge binds
to the cation-exchange column. The bound complexes are eluted using a
salt gradient flow separately from respective columns.

[0105] FIG. 4 shows the separation of ANGex from free LF, on UNO-S
cation-exchange chromatogram using NaCl gradient (60-80% B in 80 min).
Unbound (fdn)-LF (50 μM) eluted as a single peak at retention time of
123.3 min (Curve 1). The retention time of (fdn)-LF peak decreased to
121.6 min for ANGex with ANG/LF molar ratio 1.0 (Curve 2). Elution of
ANGex was monitored by measuring absorbance at 214 nm.

Example-9

Molecular Stability of ANGex

[0106] The three-dimensional structure of ANG at the physiological pH and
temperature conditions is responsible for it multi-functional activity.
Changes in milieu conditions such as the pH, ionic strength, as well as
interaction of ANG with other proteins, ligands and denaturants influence
the structure-functional properties of the native state. Higher
temperatures also lead to unfolding of the native structure leading to
diminished or total loss of ANG activity. Data on changes in the
secondary structure of ANG were obtained using Circular Dichroism (CD)
spectrophotometric assay. ANG polypeptide backbone is optically active in
the far ultraviolet region (170-250 nm) and different secondary
structures exhibit characteristic CD spectra.

[0107] In an experiment to study the acid-tolerance/resistance of ANGex, a
series of samples were prepared with 10 μM of ANGex, dissolved in 1-mL
of buffers with varying pH ranging from 2.0 to 8.0. After incubation at
37° C. for 30 min with gentle shaking, the CD spectrum of each of
the sample was recorded using a CD spectrophotometer (Jasco, Japan).
Comparison of the CD spectra of ANGex samples in the range 190-240 nm,
with that for ANGex at pH 7.2 revealed only minor changes in the
secondary structure of the complex. This observation confirmed that ANGex
is stable to changes in pH of the solution.

[0108] A similar experiment was carried out in order to assess the
thermo-stability of ANGex. A series of ANGex samples (10 μM) in 50 mM
acetate buffer, pH 5.5, were incubated at different temperatures (50, 60
and 70° C.) for various time points (5, 10, 30, and 60 min). After
incubation, the CD spectrum of each of the sample was recorded using a CD
spectrophotometer equipped with a thermostatic cell holder. There were no
significant changes in the CD profile of ANGex at higher temperatures
when compared with the profile at room temperature, confirming that ANGex
remained its native structure at elevated temperatures.

Measurement of ANG Activity by Placental RNase Inhibitor (PRI) Assay

[0109] For both acid and thermal stability testing protocols, the residual
activity of ANGex was monitored by an in vitro binding assay for ANG
using PRI (Bond M D., Anal. Biochem. 173:166-173, 1988). In brief, test
samples (volume range: 0 to 40 μL) were added to 40 μL 0.5 M Tris
buffer, pH 7.5 (with 5 mM EDTA, 10 mM DTT, 0.5 mg/mL human serum albumin)
containing of 0.53 pmol PRI (Sigma). The mixtures were made up to a final
volume of 90 μL with deionized RNase-free water, incubated for 5 min
for optimal interaction between ANG and PRI, followed by an addition of
RNase A (0.58 μmol of RNase A in 10 μL of 5 mM Tris, 0.1 mg/mL
lysozyme, pH 7.5). The reaction was initiated by the addition of 0.1-mL
of 1% yeast RNA (freshly dissolved in RNase-free water and passed through
a sterile 0.45 μm filter). After incubation for 25 min at 25°
C., the reactions were terminated with 0.2-mL ice-cold quenching reagent
(1.16 N perchloric acid with 5.9 mM uranyl acetate) and the mixture was
homogenized and placed on ice for 25 min. After centrifugation at
4° C. (15000×g for 5 min.), test samples were made from
0.2-mL aliquots diluted to 1-mL volume with 5 mM Tris, pH 7.5 containing
0.1 mg/mL lysozyme (in order to minimize adsorption to container
surfaces). The absorbance at 260 nm was recorded for each sample and
subtracted from that of a blank solution prepared in an identical manner
except that water was added instead of RNase, to give ΔA260.

[0110] A linear relationship between the RNase concentration and increased
absorption at 260 nm due to the presence of the acid-soluble nucleotides
produced from the hydrolysis of the yeast RNA, is observed. Two types of
standards were used with each binding assay, i) containing 0.58 pmol (2.9
nM) RNase A in the absence of PRI or ANG (ΔAE); and ii) containing
both the RNase A and 0.53 pmol (2.6 nM) PRI (AAEI). These standards
represent the maximum and minimum obtainable ΔA260 values,
respectively, in the absence of ANG in the sample. Increases in
ΔA260 values above the minimum are proportional to the amount
of ANG present, and the concentration is calculated using the equation:
[ANG]=[(ΔAsample-AAEI)/ΔAE]×2.9 nM. Wherein,
ΔAsample, ΔAEI and ΔAE are absorbance values of the
sample, the RNase A plus PRI standard assay, and the RNase A standard,
respectively. In particular cases, when impure samples are assayed,
concentrations are expressed in terms of RNase A equivalents in μg/mL
instead of nanomolar ANG concentration. A comparative activity of ANG,
before and after incubation at different temperatures as well as
incubation in buffers at various pH, are used in the estimation of
acid-tolerance/resistance and thermo-stability of ANGex and ANG samples.

Example-10

Measurement of functional activities of angex

RNase Activity of ANGex

[0111] RNase activity of ANG is far lower than that of RNase A
(Ribonuclease A) and therefore a highly sensitive assay was used to
measure this enzymatic activity. This assay monitors the kinetics of
cleavage of fluorescent tagged oligonucleotides (substrates) by ANGex and
measures the fluorescence emission following the cleavage (Park C et al.,
Biochemistry 41:1343-1350, 2002).

[0112] The RNase substrates are short oligonucleotides of sequences (AUAA,
AAUAAA, AAAUAAAA) each attached to a fluorophor, 6-carboxy fluorescein
(6-FAM) at the 5' end and to a quencher, 6-carboxytetramethyl-rhodamine
(6-TAMRA) at the 3' end. When the substrate is intact, 6-TAMRA quenches
the fluorescence of 6-FAM. Ribonucleolytic cleavage of the substrate
results in the fluorescence emission by 6-FAM. RNase substrate (6 nM)
solution was prepared in 1 mM Bis-Tris buffer, pH 6.0 containing 0.1M
NaCl. A 2-mL substrate solution was mixed with 50 nM ANGex and tested at
25° C. Fluorescence emission of the reaction mixture was measured
at 515 nm with excitation at 490 nm using a fluorescence
spectrophotometer (QuantaMaster 1, Photon Technology International, NJ).
Values of ΔF/Δt were determined by a linear least squares
regression analysis of the initial fluorescence (F) with time (t). This
rate was further used in the following equation:
kcat/KM={(ΔF/Δt)/(Fmax-F0)}.(1/[E]), where [E] is the
concentration of ANGex. Fmax is obtained from a separate experiment by
adding 0.1 μM RNase A to the reaction mixture and F0 is the initial
fluorescence intensity without ANG.

[0113] A comparison of the kcat/KM for (fdn)-ANG with that of ANGex
prepared from different substrates indicated the effect of
immobilization.

Antimicrobial Activity of ANGex

[0114] Microbial metabolism cause electrical charge alterations in
cultivation media due to breakdown of nutrients. A Bactometer®
Microbial Monitoring System Model-128 (bioMerieux Vitek, Hazelwood, Mo.)
was used to monitor bacterial growth by measuring impedance signals (a
function of both capacitance and conductance) in the cultivation media.
Growth Impedance Detection Assay (GIDA). Growth Impedance Detection Assay
(GIDA) was performed in 16-well modules; briefly, a volume of 0.5-mL
double-strength tryptic soy broth (2×TSB) was added to each well. A
volume of 0.25-mL of test sample (final concentration: 1 mg/mL) followed
by 0.25-mL of bacterial suspension (104 cells/mL) prepared in 0.9% saline
was added to the wells. Addition of 0.5-mL saline or bacterial suspension
to module wells with 0.5-mL TSB (2×) served as controls for
sterility and growth, respectively. The inoculated modules (final volume:
1-mL) were incubated at 37° C., and impedance changes in the media
was continuously monitored by the Bactometer® at 6-min intervals for
48-h. Bacterial growth curves were graphically displayed as percent
changes of impedance signals versus incubation time. The amount of time
required to cause a series of significant deviation from baseline
impedance value was defined as the `detection time` (DT). Difference in
DT values between growth control and test samples was considered as the
`stasis` (growth-inhibition) time.

[0115] The antimicrobial activity of ANGex (immobilized on GRP) was
compared with that of (fdn)-ANG and GRP (galactose-rich polysaccharide
from agar) activities, tested at 1 mg/mL concentration. ANG demonstrated
ability to inhibit the growth of various microbial pathogens. The
immobilization process of the free dispersed native ANG with GRP
substrate resulted in the generation of an ANGex complex with enhanced
microbial growth inhibition activity as shown in Table-1.

[0116] Ferric reducing/antioxidant power (FRAP) assay (Benzie IF, et al.,
Methods in Enzymology: Oxidants and Antioxidants, ed. L Packer, ppl 5-27,
Orlando: Academic Press, 1999) with minor modifications has been used to
measure the antioxidant activity of ANGex complexes. The FRAP reagent was
prepared by mixing 40 mL of 0.3 M acetate buffer (pH 3.6), 4 mL of 20 mM
ferric chloride, and 4 mL of 10 mM TPTZ
[2,4,6-Tris(2-pyridyl)-s-triazine]. Serial dilutions (0.1 to 1.0 mM) of
6-OH-2,5,7,8-tetramethyl chroman-2-carboxylic acid (CAS 53188-07-1) were
used as FRAP standards. All reagents were brought to 37° C. prior
to the assay. FRAP assay was performed in a 96-well microplate by mixing
20 μL of DI water, 10 μL of ANGex complex sample, and 150 μL of
FRAP reagent. In combination studies 10 μL of DI water and 20
quadraturel of ANGex were mixed with 150 μL of FRAP reagent. After
instant incubation at 37° C. for 5 min (for ascorbic acid) and for
a time lapse of 5 min to 24 h (for ANGex and milk-derived ANG) the
absorbance of reaction mixtures was measured at 593 nm (Spectramax
340PC).

[0117] The FRAP reaction kinetics (measured as the rate of increase in
absorbance of reaction mixtures at 593 nm) of ANGex was compared with its
source material, the (fdn)-ANG from bovine milk, (fdn)-LF from bovine
milk and antioxidant standards (i.e. vitamin-C, TROLOX and FeSO4).

[0118] FIG. 5A & B shows the antioxidant activity of ANGex as determined
by kinetic FRAP assay by measuring the increase in absorbance at 593 nm.
The antioxidant efficiency (r) was measured as change in absorbance with
the concentration of ANGex formed at different compositions. In FIG. 5A,
the FRAP activity of ANGex formed by the interaction of 694 μM of
(fdn)-ANG with 125 μM of (fdn)-LF, is compared with the corresponding
individual activities of the same concentration of (fdn)-LF and
(fdn)-ANG, under the same test conditions. All the systems studied show
activity in three stages, with a gradual rise in the initial 14 hours, a
steep rise in the period 14-18 hours and saturation in the final 6 of
twenty four hours. ANGex showed consistently higher absorbance throughout
the duration of the experiment. After the initial 14-h, ANGex had an
absorbance of 0.47, while (fdn)-forms of ANG and LF were at 0.29 and
0.14, respectively. After 24-h, the maximum absorbance at 593 nm,
attained by (fdn)-LF and (fdn)-ANG are 0.87 and 0.64, respectively,
whereas ANGex demonstrated a maximum absorbance of 1.18, which is a 35%
increase from that of (fdn)-LF and a 84% increase from that of (fdn)-ANG.
FIG. 5B shows that when individually tested, (fdn)-ANG demonstrated
antioxidant efficiency (r) of 0.65 Abs/mM. When complexed with 25 μM
of LF, the `r` value increased to 0.67, indicating an enhancement of the
antioxidant power. At the same, ANG complexed with higher concentrations,
75 and 125 μM of LF showed significantly reduced `r` values of 0.3 and
0.38, respectively.

DEFINITION OF TERMS

[0119] Angiogenic activity is the chemical stimulation of hemovascular
development in tissue. It is generally associated with diffusible
substances produced by a variety of cell types.

[0120] Angiogenin: As used herein, "angiogenin" or "ANG" refers to an
angiogenic-stimulating factor, which is also 14-kDa heparin-binding
protein that occurs in most cells, also present in various biological
fluids such as plasma and milk.

[0125] Lactoferrin (LF): As used herein, "lactoferrin", or "LF" refers to
various protein preparations and forms, including but not limited to,
lactoferrin-(tcr) (as described in Naidu U.S. Pat. No. 7,125,983),
freely-dispersed native (fdn)-lactoferrin which includes metal-saturated
(holo), partially saturated and metal-free (apo) forms of LF. The
LF-bound metal is preferably copper, and other bound metals include zinc,
iron, manganese, chromium, aluminum and gallium. The term LF further
refers to fully and partially glycosylated polypeptide chains of LF,
incomplete polypeptide chains including half-molecules comprising C- and
N-terminus lobes of LF. The term LF categorically excludes aggregated-LF
and immobilized (Im)-LF forms (as described in Naidu U.S. Pat. No.
6,172,040 B1, issued Jan. 9, 2001) that are devoid of any (fdn)-LF.

[0126] Mitogenic activity is the chemical stimulation of cell division.

[0127] Ribonuclease (RNAse) activity is characterized by the degradation
of large RNA molecules, such as the 28S and 18S ribosomal RNAs, to lower
molecular weight species.

[0128] Freely-dispersed native (fdn): As used herein, "freely-dispersed
native" (fdn) refers to isolated protein molecules free of
auto-aggregation or polymerization and free from binding or
immobilization to other substrates.

[0129] It will be understood by those of skill in the art that numerous
and various modifications can be made without departing from the spirit
of the present invention. Therefore, it should be clearly understood that
the forms of the present invention are illustrative only and are not
intended to limit the scope of the present invention.